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A method for rapidly preparing sulfhydryl-modified DNA nano-gold complexes (DNA-AUNP)

A sulfhydryl-modified, nano-gold technology, used in DNA/RNA fragments, DNA preparation, recombinant DNA technology, etc., can solve the problems of inability to form hydrogen bonds and low loading of DNA probes, and achieve faster uploading rate and reduce DNA consumption. Effect

Active Publication Date: 2020-12-22
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the rapid preparation of DNA-AuNPs under low pH conditions is only suitable for the uploading of linear DNA on gold nanoparticles due to the inability to form hydrogen bonds under low pH conditions.
In 2009, Fan Chunhai's research group successfully prepared the molecular beacon-AuNP complex by using the method of mixing and coating with short-chain auxiliary probes, but the loading capacity of DNA probes was relatively low (Angew.Chem.Int.Ed. 2009, 48(46), 8670-8674)
Therefore, it is very necessary to find a preparation method that can be fast, stable, efficient, quantitative and does not interfere with downstream applications to solve the problem of uploading DNA with secondary structure on the surface of gold nanoparticles

Method used

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  • A method for rapidly preparing sulfhydryl-modified DNA nano-gold complexes (DNA-AUNP)
  • A method for rapidly preparing sulfhydryl-modified DNA nano-gold complexes (DNA-AUNP)
  • A method for rapidly preparing sulfhydryl-modified DNA nano-gold complexes (DNA-AUNP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Real-time monitoring of sulfhydryl-modified DNA with spacer chains of the same length and different composition by dynamic light scattering. The particle size change of DNA-AuNP when DNA-AuNP is prepared according to the "aging-salting" method.

[0038] A linear DNA (15-A 10 -SH, 15-T 10 -SH, 15-EG 18 -SH, Table 1) were mixed with 40mM dithiothreitol (DTT) and 2% (V / V) triethylamine (TEA) solution, incubated at room temperature for 30 minutes, and the mixture was carried out twice using NAP-5 column For secondary purification, DNA was eluted in 10 mM phosphate buffered saline (pH 7.4). The purified DNA was added to gold nanoparticles with a diameter of 13nm prepared by the citric acid reduction method, and the ratio of the input substance was 500:1 (DNA:AuNP). According to the "aging-salting" method, the three steps of aging, adding salt, and incubating are used to prepare DNA-AuNP. The aging process is 18 hours after the addition of nano-gold to the DNA,...

Embodiment 2

[0040] Example 2. Determination of the coating amount and particle size of thiol-modified DNA with different lengths of OEG spacer chains on gold nanoparticles at different salt concentrations during the 2-hour aging process.

[0041] DNA was reduced and purified according to the method in Example 1. In this example, 15-EG 6 -SH, 15-EG 12 -SH, 15-EG 18 -SH was used as the DNA of the spacer strand for the preparation of DNA-AuNPs. A 2-hour incubation of DNA with AuNPs was performed in phosphate buffered saline containing different salt concentrations. The change in particle size of DNA-AuNPs was measured using dynamic light scattering during the incubation. After the incubation, take pictures, perform UV-visible spectrum test, and then perform centrifugation at 15000rpm, take 100 μL of supernatant, quantitatively measure the probe concentration in the supernatant according to UV-visible spectrum and calculate the coating amount.

[0042]The results show that OEG as a space...

Embodiment 3

[0043] Example 3. Determination of the ratio of the amount of different substances, 15-EG 18 -The DNA-AuNP particle size change curve with time during the 2-hour aging incubation of SH and 13nm gold nanoparticles and the upload amount after 2 hours of incubation.

[0044] Reduction and purification of 15-EG according to the method in Example 1 18 -sh. Detection of 15-EG in the proportion of different substances input by DLS 18 -SH and nano-gold change in particle size during the 2-hour aging process, and the ratios of the substances are 100:1, 200:1, 300:1, 400:1, 500:1, and the selection time is 0, 0.5, 1 , 1.5, 2h when the point of particle size measurement. After aging for 2 hours, the sample was centrifuged at 15,000 rpm, and 100 μL of the supernatant was taken, and the concentration of the probe in the supernatant was quantitatively determined according to ultraviolet-visible spectroscopy and the coating amount was calculated.

[0045] The results showed that with the...

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Abstract

The present invention relates to a method for rapidly preparing a thiol-modified DNA nanometer gold complex (DNA-AuNP), wherein DNA is modified with thiol using different lengths of oligoethylene glycol (OEG) separation chains as separation chains so as to perform the rapid preparation of DNA-AuNP. According to the present invention, the loading rate of the DNA on the nanometer gold surface can be significantly improved, and the loading rate is increased along with the increase of the salt concentration; the method is the general DNA-AuNP preparation method, and is suitable for the DNA having different thiol modification (5' or 3') positions, different lengths and different sequences; the method can be used for loading linear single-stranded DNA on the nanometer gold and loading DNA probes with a secondary structure on the nanometer gold surface, wherein the DNA probe (such as molecular beacon) with a secondary structure can be directly loaded on the nanometer gold surface in one step; and the method is suitable for different sizes of nanometer gold, especially for the large-size nanometer gold with relatively poor stability.

Description

technical field [0001] The invention relates to a method for rapidly preparing sulfhydryl-modified DNA nano-gold complex (DNA-AuNP) by using thiol-DNA modified by oligoethylene glycol spacer (OEG), and belongs to the field of biotechnology. Background technique [0002] Sulfhydryl-modified DNA nano-gold complex (DNA-AuNP) has very important applications in the fields of nano-self-assembly structure construction, biosensing technology and drug delivery. Efficient molecular recognition of DNA on gold nanomaterial interfaces is fundamental for these applications. Extensive studies have shown that the number, density, conformation, composition of the protective layer, shape and size of the composite probe and many other factors of DNA on DNA-AuNPs significantly affect the efficiency of molecular recognition. To solve the technical problem of poor reproducibility of DNA-AuNP-based applications, the key is the preparation of DNA-AuNP, and the quantity and conformation of DNA on t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6811C12N15/11C12N15/10
CPCC12Q1/6811C12Q2563/137C12Q2563/155
Inventor 娄新徽徐擎
Owner CAPITAL NORMAL UNIVERSITY
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